Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. VI. Experimental disturbance of antennal branch formation

In the male silkmoth Antheraea polyphemus, the formation of the side branches of the quadripectinate antennal flagellum was disturbed by an experimental manipulation. Normally the side branches develop in the pupa via deep incisions which proceed from the periphery towards the centerline of the leaf-shaped antennal anlage. Local removal of the uppermost, pigmented cuticular layers of the pupal antennal pocket ('cuticular window') led to a local standstill of branch formation in the manipulated region of the pocket, most probably caused by increased evaporation of water through the remaining layers of meso- and endocuticle. These parts of the antenna retained an unbranched, plate-like shape. This early morphogenetic stage was conserved by the secretion of antennal cuticle. Besides cuticle formation, development of sensilla is not impeded by the manipulation. In the plate-shaped regions, the initial pattern formed by the sensilla in the antennal epidermis is preserved, because they maturate at their birthplaces. In the individual segments, the pattern of sensilla shows a mirror-like symmetry with respect to the segmental midline. From the edge to the midline, we found large s. trichodea, followed by small s. trichodea, s. basiconica, and s. coeloconica on the dorsal side whereas on the ventral side, there are only large s. trichodea and s. campaniformia. We conclude that the development of the featherlike antennal shape on the one hand and the development of sensilla and cuticle on the other hand are independent processes.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. V. Development of the peripheral nervous system

The imaginal antenna of the male silkmoth Antheraea polyphemus is a feather-shaped structure consisting of about 30 flagellomeres, each of which gives off two pairs of side branches. During the pupal stage (lasting for 3 weeks), the antenna develops from a leaf-shaped, flattened epidermal sac ('antennal blade') via two series of incisions which proceed from the periphery towards the prospective antennal stem. The development of the peripheral nervous system was studied by staining the neurons with an antibody against horseradish peroxidase as well as by electron microscopy. The epithelium is subdivided in segmentally arranged sensillogenic regions alternating with non-sensillogenic regions. Immediately after apolysis, clusters consisting of 5 sensory neurons each and belonging to the prospective sensilla chaetica can be localized at the periphery of the antennal blade in the sensillogenic regions. During the first day following apolysis, the primordia of ca. 70 000 olfactory sensilla arise in the sensillogenic regions. Axons from their neurons are collected in segmentally arranged nerves which run towards the CNS along the dorsal as well as the ventral epidermis and are enveloped by a glial sheath. This 'primary innervation pattern' is completed within the second day after apolysis. A first wave of incisions ('primary incisions') subdivide the antennal blade into segmental 'double branches' without disturbing the innervation pattern. Then a second wave of incisions ('secondary incisions') splits the double branches into single antennal branches. During this process, the segmental nerves and their glial sheaths are disintegrated. The axons are then redistributed into single branch nerves while their glial sheath is reconstituted, forming the 'secondary', or adult, innervation pattern. The epidermis is backed by a basal lamina which is degraded after outgrowth of the axons, but is reconstituted after formation of the single antennal branches.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. I. The leaf-shaped antenna of the pupa from diapause to apolysis

The antenna of the male silkmoth Antheraea polyphemus is a featherlike structure consisting of a central stem and ca. 120 side branches, which altogether carry about 70,000 olfactory sensilla. We investigate the development during the pupal phase. At the end of diapause, the antennal rudiment consists of a leaf-shaped, one-layered epidermal sac. It is supplied with oxygen via a central main trachea, which gives off numerous thin side branches. These are segmentally arranged into bundles which run to the periphery of the antennal blade. When the epidermis retracts from the pupal cuticle (apolysis; stage 1), it consists of cells which are morphologically uniform. The epidermal cells form a network of long, irregular basal protrusions (epidermal feet), which crisscross the antennal lumen. During the first day post-apolysis (stage 2), the antennal epidermis differentiates into alternating thick 'sensillogenic' and thin 'non-sensillogenic' areas arranged in stripes which run in parallel to the tracheal bundles. Numerous dark, elongated cells, which might be the sensillar stem cells, are scattered in the sensillogenic epithelium. A number of very early sensilla has been found at the distal edges of the sensillogenic stripes in positions which later will be occupied by sensilla chaetica. The whole antennal blade is enveloped by the transparent ecdysial membrane, consisting of the innermost layers of the pupal cuticle which are detached during apolysis.     

Morphogenesis of the antenna of the male silkmoth. Antheraea polyphemus, III. Development of olfactory sensilla and the properties of hair-forming cells

During adult development of the male silkmoth Antheraea polyphemus, the anlagen of olfactory sensilla arise within the first 2 days post-apolysis in the antennal epidermis (stage 1-3). Approximately on the second day, the primary dendrites as well as the axons grow out from the sensory neurons (stage 4). The trichogen cells start to grow apical processes approximately on the third day, and these hair-forming 'sprouts' reach their definite length around the ninth day (stages 5-6). Then the secretion of cuticle begins, the cuticulin layer having formed on day 10 (stage 7a). The primary dendrites are shed, the inner dendritic segments as well as the thecogen cells retract from the prospective hair bases, and the inner tormogen cells degenerate around days 10/11 (stage 7b). The hair shafts of the basiconic sensilla are completed around days 12/13 (stage 7c), and those of the trichoid sensilla around days 14/15 (stage 7d). The trichogen sprouts retract from the hairs after having finished cuticle formation, and the outer dendritic segments grow out into the hairs: in the basiconic sensilla directly through, and in the trichoid sensilla alongside, the sprouts. The trichogen sprouts contain numerous parallel-running microtubules. Besides their cytoskeletal function, these are most probably involved in the transport of membrane vesicles. During the phase of cuticle deposition, large numbers of vesicles are transported anterogradely from the cell bodies into the sprouts, where they fuse with the apical cell membrane and release their electron-dense contents (most probably cuticle precursors) to the outside. As the cuticle grows in thickness, the surface area of the sprouts is reduced by endocytosis of coated vesicles. When finally the sprouts retract from the completed hairs, the number of endocytotic vesicles is further increased and numerous membrane cisterns seem to be transported retrogradely along the microtubules to the cell bodies. Here the membrane material will most probably be used again in the formation of the sensillum lymph cavities. Thus, the trichogen cells are characterized by an intensive membrane recycling. The sensillum lymph cavities develop between days 16-20 (stage 8), mainly via apical invaginations of the trichogen cells. The imago emerges on day 21.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. II. Differential mitoses of 'dark' precursor cells create the Anlagen of sensilla

The antenna of the male silkmoth Antheraea polyphemus develops from a one-layered, flattened epidermal sac during the pupal phase. Within the first day post-apolysis (developmental stages 1 and 2), this epithelium differentiates into 'sensillogenic' and 'nonsensillogenic' regions, while numerous slender 'dark cells' interpreted as the precursor cells of sensilla arise in the former. Approximately between the first and second day post-apolysis (developmental stage 3), the dark cells retract to the apical pole of the epidermis, assume a round shape, and undergo a series of differential mitoses with spindles usually oriented parallel to the epidermal surface. These mitoses finally yield the Anlagen of the olfactory sensilla trichodea, each consisting of mostly 6-7 dark cells arranged side by side. In most of the Anlagen, 3-4 of these cells are situated more basally, each giving off a slender apical process which together are arranged in a fascicle. These are the prospective 2-3 sensory neurons plus the thecogen cell, which most probably is a sister cell of the former. Three additional cells are arranged more apically and partly enclose the fascicle of presumed sensory and thecogen cell processes. These are interpreted as the trichogen plus 2 tormogen cells, one of the latter degenerating later during development. In the basal region of the sensillogenic epidermis, massive signs of cell degeneration have been found. At stage 3, the basal epidermal feet in the non-sensillogenic regions have assumed a more uniform orientation as compared with the preceding stages.     

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.     

Reconstruction and morphometry of silkmoth olfactory hairs: A comparative study of sensilla trichodea on the antennae of male Antheraea polyphemus and Antheraea pernyi (Insecta,Lepidoptera)

Summary Olfactory trichoid hairs on the antennae of male Antheraea silkmoths were reconstructed with respect to the following parameters: number, shape, course, and dimensions of outer dendritic segments as well as the numbers of their microtubules; inner and outer dimensions of the cuticular hair shafts; and number and distribution of pores and pore tubules in the hair walls. The smallest distances between dendritic membranes and inner hair surfaces were determined with respect to the possibility of pore tubule contacts. It was shown that most hairs contain one thick and one, or frequently two, thin dendrites. The number of microtubules in the dendrites is correlated with dendrite diameter, which decreases towards the hair tip. The dendrites form numerous swellings and constrictions: this beading occurs especially along the thin dendrites. The dendrites do not run straight, but rather follow a sinuous course in the hairs. The density of wall pores is lowest in the basal region of the hairs. Only in relatively few places do the dendritic membranes get near enough the hair walls to come into the probable range of the pore tubules. In the sensilla trichodea of A. polyphemus, the hairs as well as the dendrites have markedly smaller diameters than in A. pernyi.     

Sequence complexities of mRNA populations during adult wing development in the silkmoth Antheraea polyphemus

The total poly A⁺ messenger RNAs of epidermal cells in the wing sac of the silkmoth Antheraea polyphemus as a function of development (which is under the influence of the moulting hormone, 20-hydroxyecdysone) has been analyzed by cDNA-mRNA hybridizations. Kinetic and numerical analysis of homologous hybridizations indicate the messenger populations to be made up of several kinetic classes, the total complexity being similar at all stages of development. Heterologous hybridizations indicate clear differences between two stages: (i) at the beginning of development characterized by growth and development and (ii) at the end of development characterized by differentiation and cuticular secretion. The principal difference between the two stages has been shown, by isolation and characterization of null cDNA (cDNA enriched for the second stage) to be due to the appearance of several hundred new mRNAs belonging to the moderately abundant class at the second stage. Analysis of translation products by SDS-PAGE and immunoprecipitation confirm and extend these results.     

A receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus

Male moths respond to conspecific female-released pheromones with remarkable sensitivity and specificity, due to highly specialized chemosensory neurons in their antennae. In Antheraea silkmoths, three types of sensory neurons have been described, each responsive to one of three pheromone components. Since also three different pheromone binding proteins (PBPs) have been identified, the antenna of Antheraea seems to provide a unique model system for detailed analyzes of the interplay between the various elements underlying pheromone reception. Efforts to identify pheromone receptors of Antheraea polyphemus have led to the identification of a candidate pheromone receptor (ApolOR1). This receptor was found predominantly expressed in male antennae, specifically in neurons located beneath pheromone-sensitive sensilla trichodea. The ApolOR1-expressing cells were found to be surrounded by supporting cells co-expressing all three ApolPBPs. The response spectrum of ApolOR1 was assessed by means of calcium imaging using HEK293-cells stably expressing the receptor. It was found that at nanomolar concentrations ApolOR1-cells responded to all three pheromones when the compounds were solubilized by DMSO and also when DMSO was substituted by one of the three PBPs. However, at picomolar concentrations, cells responded only in the presence of the subtype ApolPBP2 and the pheromone (E,Z)-6,11-hexadecadienal. These results are indicative of a specific interplay of a distinct pheromone component with an appropriate binding protein and its related receptor subtype, which may be considered as basis for the remarkable sensitivity and specificity of the pheromone detection system.     

Diffusion barriers in silkmoth sensory epithelia: application of lanthanum tracer to olfactory sensilla of Antheraea polyphemus and Bombyx mori

The distribution of diffusion barriers in silkmoth olfactory sensilla has been investigated with ionic lanthanum. The tracer was applied from the apical side of the sensory epithelium by first pinching off the hair tips and then dipping the antennal branches into the La(NO(3))(3) solution. The tracer neither passed the apical septate junctions between the dendrite and the thecogen cell nor those between thecogen, trichogen, and tormogen cells, nor the tight contact between the apical membrane of the tormogen cell and the cuticle. After perfusing the hemolymph space with La(NO(3))(3) solution, the tracer was found in the clefts between the thecogen, trichogen, tormogen, and epidermis cells, but not in those between the receptor cells and the thecogen cell, or between the axon and the glial envelope. Lanthanum neither entered the receptor-lymph space nor the subcuticular space. Therefore, (i) receptor-lymph space, subcuticular space, and hemolymph space are isolated from each other, and (ii) the cleft between thecogen and sensory cell is separated from the hemolymph as well as from the receptor-lymph spaces. Furthermore, the results indicate that pleated septate junctions form the diffusion barriers in silkmoth olfactory sensilla.     

Dynamics of 'immotile' olfactory cilia in the silkmoth Antheraea

Living olfactory sensory dendrites of the silkmoth Antheraea which are modified cilia lacking the central microtubule pair have been observed by means of video microscopy in sensilla from which the apical tips had been pinched off as well as in vitro after isolation. Dendrites project out of the opened hair tips cither spontaneously without manipulation or after application of basal pressure via a syringe connected to the haemolymph side. Spontaneously appearing dendrites can repeatedly project up to ca. 60 mum from, and retract back into, the hairs. They tend to remain straight, but curve if they project too far and bend on meeting an obstacle. The average elongation velocity of the dendrites is 0.4 mum/sec. After application of basal pressure, large numbers of dendrites immediately slide out of the apically opened hairs. These dendrites usually detach at their bases and float free in the solution until settling down at the bottom of the petri dish. They are able to make active movements, for example bending between points of attachment. Dendrites tend to adhere to other dendrites, sometimes making sliding movements against each other. The ciliary olfactory dendrites are backed by a large number of microtubules which appear to be interconnected by fine filaments, most probably microtubule-associated proteins (MAPs). The elongation and shortening of the dendrites is explained here by a sliding-filament mechanism similar to the one acting in 'true motile' cilia. As the cytoskcleton is not as highly organized as in the latter, the resulting movements are limited to elongation and contraction, bending being brought about only passively by apical resistance. Membrane beads have been observed to appear on, and move along, the dendrites. Their number increases with the age of the preparation.     

Atomic-force microscopy on the olfactory dendrites of the silkmoths Antheraea polyphemus and A. pernyi

Olfactory transduction is thought to occur in the outer dendritic membrane of insect olfactory receptor neurons. Electrophysiological studies have indicated that the outer dendritic membrane has non-specific cation channels and inositol-triphosphate-dependent Ca²⁺ channels. The presence of such channels is further supported by the observation that pheromone-stimulated dendrites take up cobalt. However, to date, there is no structural evidence for these channels. Therefore, in order to search for putative ion channels, we have imaged the membrane of the olfactory dendrites in the scanning electron microscope (SEM) and the atomic-force microscope (AFM), after extruding the dendrites out of the olfactory hairs and fixing them on plastic coverslips. With the aid of the SEM, we could see the beaded structure of the dendrite but no fine structural details, as the membrane was sputtered with gold. With the use of the contact mode of the AFM, we could see “pores” that were deeper than 3 nm and with a diameter of about 15 nm. The density of the “pores” was approximately 20/µm² or 10?000 pores per thick dendrite. We believe these to be putative ion channels based on indirect evidence.     

Morphogenesis of silkmoth chorion: Initial framework formation and its relation to synthesis of specific proteins

Morphogenesis of the silkmoth eggshell is described at the ultrastructural level. Four zones are each assembled in a distinct manner and during a distinct developmental period: the innermost vitelline membrane and the adjacent trabecular layer appear consecutively, followed by a thin sieve layer, and a thick, lamellate chorion. Once formed, the sieve layer remains attached to microvilli, and thus all components which assemble into lamellae must pass through the sieve layer. Initially, lamellogenesis (and sieve layer formation) occurs in patches overlying trabeculae. Lamellae quickly fuse and new ones are added, presumably by apposition. Distinct types of lamellae seen in the mature chorion are already distinguishable in early lamellogenesis. The final lamellar number is attained before the developing chorion is one-half its final thickness or one-fifth its final dry weight. The early lamellae constitute a framework which is subsequently modified through expansion and densification. Proteins which may represent components of various parts of the eggshell have been identified on the basis of their timing of synthesis, relative amino acid compositions, and spatial distributions within the chorion.     

Antennal-specific pheromone-degrading aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori

Female moths produce blends of odorant chemicals, called pheromones. These precise chemical mixtures both attract males and elicit appropriate mating behaviors. To locate females, male moths must rapidly detect changes in environmental pheromone concentration. Therefore, the regulation of pheromone concentration within antennae, their chief organ of smell, is important. We describe antennal-specific aldehyde oxidases from the moths Antheraea polyphemus and Bombyx mori that are capable of catabolizing long chain, unsaturated aldehydes such as their aldehyde pheromones. These soluble enzymes are associated uniquely with male and female antennae and have molecular masses of 175 and 130 kDa, respectively. The A. polyphemus aldehyde oxidase has been localized to the olfactory sensilla which contain the pheromone receptor cell dendrites. These same sensilla contain a previously described sensilla-specific esterase that degrades the acetate ester component of A. polyphemus pheromone. We propose that sensillar pheromone-degrading enzymes modulate pheromone concentration in the receptor space and hence play a dynamic role in the pheromone-mediated reproductive behaviors of these animals.     

Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera)

Antheraea assama, an economically important and scientifically unexplored Indian wild silkmoth, is unique among saturniid moths. For this species, a total of 87 microsatellite markers was derived from 35 000 expressed sequence tags and a microsatellite‐enriched sub‐genomic library. Forty individuals collected from Tura and West Garo Hills region of Northeast India were screened for each of these loci. Ten loci from expressed sequence tags and one from genomic library were found to be polymorphic. These microsatellite markers will be useful resources for population genetic studies of A. assama and other closely related species of saturniids. This is the first report on development of microsatellite markers for any saturniid species.